T-lymphocytes play a major role in the immune response by interacting with target and antigen presenting cells. For example, the T-lymphocyte mediated killing of target cells is a multi-step process involving adhesion of a cytolytic T-lymphocyte to a target cell. And, helper T-lymphocytes initiate the immune response by adhesion to antigen-presenting cells.
These interactions of T-lymphocytes with target and antigen-presenting cells are highly specific and depend on the recognition of an antigen on the target or antigen-presenting cell by one of the many specific antigen receptors on the surface of the T-lymphocyte.
The receptor-antigen interaction of T-lymphocytes and other cells is also facilitated by various T-lymphocyte surface proteins, e.g., the antigen receptor complex CD3(T3) and accessory molecules CD4, LFA-1, CD8, and CD2. It is also dependent on accessory molecules, such as LFA-3, ICAM-1 and MHC that are expressed on the surface of the target or antigen-presenting cells. In fact, it is hypothesized that the accessory molecules on the T-lymphocytes and on the target or antigen-presenting cells interact with each other to mediate intercellular adhesion. Accordingly, these accessory molecules are thought to enhance the efficiency of lymphocyte-antigen-presenting cell and lymphocyte-target cell interactions and to be important in leukocyte-endo-thelial cell interactions and lymphocyte recirculation.
For example, recent studies have suggested that there is a specific interaction between CD2 (a T-lymphocyte accessory molecule) and LFA-3 (a target cell accessory molecule) that mediates T-lymphocyte adhesion to the target cell. This adhesion is essential to the initiation of the T-lymphocyte functional response (M. L. Dustin et al., "Purified Lymphocyte Function-Associated Antigen-3 Binds To CD2 And Mediates T Lymphocyte Adhesion", J. Exp. Med., 165, pp. 677-92 (1987); Springer et al., "The Lymphocyte Function-Associated LFA-1, CD2, and LFA-3 Molecules: Cell Adhesion Receptors Of The Immune System", Ann. Rev. Immunol., 5, pp. 223-52 (1987)). And, monoclonal antibodies to either LFA-3 or CD2 have been shown to inhibit a spectrum of cytolytic T lymphocyte and helper T lymphocyte dependent responses (F. Sanchez-Madrid et al., "Three Distinct Antigens Associated With Human T-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2, And LFA-3", Proc. Natl. Acad. Sci. USA, 79, pp. 7489-93 (1982)).
LFA-3 is found on antigen-presenting cells, and target cells, specifically on monocytes, granulocytes, CTL's, B-lymphoblastoid cells, smooth muscle cells, vascular endothelial cells, and fibroblasts (Springer et al., supra). LFA-3 exists as two distinct cell surface forms (Dustin et al., "Anchoring Mechanisms For LFA-3 Cell Adhesion Glycoprotein At Membrane Surface", Nature, 329, pp. 846-848 (1987)). These forms differ mainly by their mechanism of attachment to lipid bilayers of biological membranes. One such anchoring mechanism is via a stretch of hydrophobic amino acids, also referred to as a transmembrane domain, which penetrates the lipid bilayers. cDNA encoding this form of LFA-3, also referred to as an integrated membrane form, has been cloned and sequenced (B. Wallner et al., J.Exp.Med., 166, pp. 923-32 (1987)).
Alternatively, LFA-3 has been reported to insert into the membrane of B-lymphoblastoid cells via a phosphatidylinositol ("PI")-containing glycolipid covalently attached to the C-terminus of the protein (Dustin et al., supra). Membrane insertion of this type was deduced by observing the presence of protein after adding to the cell surface phosphatidylinositol-specific phospholipase. This enzyme releases only the PI-linked form of proteins. It does not affect the integrated membrane form. Thus, the release of LFA-3 in the presence of this enzyme suggests that LFA-3 has a PI-linked form.
The PI-linked form of LFA-3 is believed to be derived from alternative RNA splicing of a gene transcript. It appears to be selectively expressed in different cell types, and during different stages of development than the transmembrane form of LFA-3.
It would be desirable to obtain large amounts of a recombinant PI-linked form of LFA-3, than would be available from purification from natural sources, e.g. lymphoblastoid cells. More desirable would be to obtain large amounts of soluble LFA-3 from a PI-linked form of LFA-3.